Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/08—Bronchodilators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives

Definitions

• Vasoactive intestinal peptide was first discovered, isolated and purified from porcine intestine. [U.S. Pat. No. 3,879,371]. The peptide has twenty-eight (28) amino acids and bears extensive homology to secretin and glucagon. [Carlquist et al., Horm. Metab. Res., 14, 28-29 (1982)].
• VIP is known to exhibit a wide range of biological activities throughout the gastrointestinal tract and circulatory system. In light of its similarity to gastrointestinal hormones, VIP has been found to stimulate pancreatic and biliary secretion, hepatic glycogenolysis, glucagon and insulin secretion and to activate pancreatic bicarbonate release. [Kerrins, C. and Said, S. L, Proc. Soc. Exp. Biol. Med., 142, 1014-1017 (1972); Domschke, S. et al., Gastroenterology, 73, 478-480 (1977)].
• VPAC1 and VPAC2 Two types of VIP receptors are known and have been cloned from human, rat, mouse, chicken, fish and frog. They are currently identified as VPAC1 and VPAC2 and respond to native VIP with comparable affinity.
• VPAC2 receptor mRNA is found in the human respiratory tract including tracheal and bronchial epithelium, glandular and immune cells, alveolar walls and macrophages. [Groneberg et al, Lab. Invest., 81, 749-755 (2001) and Laburthe et al., Receptors and Channels, 8, 137-153 (2002)].
• VIP has also been found to stimulate renin release from the kidney in vivo and in vitro. [Porter, J. P., et al., Neuroendocrinology, 36, 404-408 (1983)]. VIP has been found to be present in nerves and nerve terminals in the airways of various animal species and man. [Dey, R. D., and Said, S. I., Fed. Proc., 39, 1062 (1980); Said, S. L, et al., Ann. N.Y. Acad. Sci., 221, 103-114, (1974)].
• VIP cardiovascular and bronchopulmonary effects are of interest as VIP has been found to be a powerful vasodilator and potent smooth muscle relaxant, acting on peripheral, pulmonary, and coronary vascular beds. [Said, S. L, et al., Clin. Res., 20, 29 (1972)]. VIP has been found to have a vasodilatory effect on cerebral blood vessels. [Lee, T. J. and Berszin, I., Science, 224, 898-900 (1984)]. In vitro studies have demonstrated that vasoactive intestinal peptide, applied exogenously to cerebral arteries, induced vasodilation, suggesting VIP as a possible transmitter for cerebral vasodilation. [Lee, T.
• Emphysema is an underlying pathology of COPD by damaging lung tissue with enlargement of the airspaces and loss of alveolar surface area. Lung damage is caused by weakening and breaking the air sacs within the lungs. Natural elasticity of the lung tissue is also lost, leading to overstretching and rupture. Smaller bronchial tubes may be damaged which can cause them to collapse and obstruct airflow, leading to shortage of breath.
• the peptide has been the target of several reported synthetic programs with the goal of enhancing one or more of the properties of this molecule.
• Takeyama et al. have reported a VIP analog having a glutamic acid substituted for aspartic acid at position 8. This compound was found to be less potent than native VIP. [Chem. Pharm. Bull., 28, 2265-2269 (1980)].
• Wendlberger et al. have disclosed the preparation of a VIP analog having a norleucine substituted at position 17 for methionine. [Peptide. Proc. 16th Eur. Pept. Symp., 290-295 (1980)].
• the peptide was found to be equipotent to native VIP for its ability to displace radioiodinated VIP from liver membrane preparations.
• Watts and Wooton have reported a series of linear and cyclic VIP fragments, containing between six and twelve residues from the native sequence. [Eur. Pat. Nos. 184309 and 325044; U.S. Pat. Nos. 4,737,487 and 4,866,039].
• Turner et al have reported that the fragment VIP(10-28) is an antagonist to VIP. [Peptides, 7, 849-854 (1986)].
• the substituted analog [4-Cl-D-Phe 6 ,Leu 17 ]-VIP has also been reported to bind to the VIP receptor and antagonize the activity of VIP.
• Gourlet et al have reported an [Arg 16 ]-VIP derivative with affinity for VIP receptors [Gourlet et al, Biochim. Biophys. Acta, 1314, 267-273 (1996)]. Onoue et al have reported a series of arginine derivatives and truncations of VIP [Onoue et al, Life Sci., 74, 1465-77 (2004) and Ohmori et al, Regul. Pept., 123, 201-7 (2004)]. A series of poly-alanine derivatives has also been reported [Igarashi et al, J. Pharm. Exper. Ther., 303, 445-60 (2002) and Igarashi et al, J. Pharm. Exper. Ther., 315, 370-81 (2005)].
• Analogs of VIP having selective VPAC1 agonist activity have been reported [Pan and Roczniak, US20050203009].
• Analogs of VIP and C-terminal pegylated derivatives have been reported has being of utility for the treatment of metabolic disorders including diabetes [Froland et al, WO2004006839, Clairmont et al, WO2005072385, Whelan et al, WO2005123109, Bokvist et al, WO2005113593 and WO2005113594, and Nestor, US20060079456 and WO2006042152].
• Native VIP has been reported to be of utility for the treatment of COPD, pulmonary hypertension and other airway disorders [WO03061680, WO0243746 and WO2005014030].
• the present invention comprises a VPAC-2 receptor agonist of the formula (I): or a pharmaceutically acceptable salt thereof. Underlined residues indicate a side-chain to side-chain covalent linkage of the first and last amino acids within the segment.
• the present invention also encompasses pharmaceutical compositions containing such agonists, and the use of such agonists for the treatment of pulmonary diseases including COPD.
• the present invention comprises a VPAC-2 receptor agonist of the formula (I): wherein: X is a hydrogen of the N-terminal amino of Histidine which may be optionally replaced by a hydrolyzable amino protecting group, most preferably by an acetyl group, Y is the hydroxy of the C-terminal carboxy of Threonine which may be optionally replaced by a hydrolyzable carboxy protecting group, most preferably by NH 2 , underlined residues indicates a side-chain to side-chain covalent linkage of the first (Lys 21 ) and last (Asp 25 ) amino acids within the segment, R 2 is Ser or Ala, R 5 is Thr, Ser, Asp, Gln, Pro or C ⁇ MeVal, R 16 is Gln, Ala, or Arg, R 18 is Ala, Lys or Glu, R 27 is Lys or Leu except that R 27 must be Lys when R 5 is C ⁇ MeVal and R 16 is Arg, R 28 is Lys or As
• Nle Norleucine
• hydrolyzable amino protecting group and “hydrolyzable carboxy protecting group”, any conventional protecting groups which can be removed by hydrolysis can be utilized in accordance with this invention. Examples of such groups appear hereinafter.
• Preferred amino protecting groups are acyl groups of the formula wherein X 3 is lower alkyl or halo lower alkyl. Of these protecting groups, those wherein X 3 is C 1-3 alkyl or halo C 1-3 alkyl are especially preferred.
• Preferred carboxy protecting groups are lower alkyl esters, NH 2 and lower alkyl amides, with C 1-3 alkyl esters, NH 2 and C 1-3 alkyl amides being especially preferred.
• alkyl means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl)hydrocarbyl radical which may be substituted or unsubstituted.
• the alkyl group is preferably C 3 to C 12 , more preferably C 5 to C 10 , more preferably C 5 to C 7 .
• the alkyl group is preferably C 1 to C 10 , more preferably C 1 to C 6 , more preferably methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, isobutyl or tertiary-butyl) or pentyl (including n-pentyl and isopentyl), more preferably methyl.
• lower alkyl means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl)hydrocarbyl radical wherein said cyclic lower alkyl group is C 5 , C 6 or C 7 , and wherein said acyclic lower alkyl group is C 1 , C 2 , C 3 or C 4 , and is preferably selected from methyl, ethyl, propyl (n-propyl or isopropyl) or butyl (n-butyl, isobutyl or tertiary-butyl).
• acyl means an optionally substituted alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl group bound via a carbonyl group and includes groups such as acetyl, propionyl, benzoyl, 3-pyridinylcarbonyl, 2-morpholinocarbonyl, 4-hydroxybutanoyl, 4-fluorobenzoyl, 2-naphthoyl, 2-phenylacetyl, 2-methoxyacetyl and the like.
• aryl means a substituted or unsubstituted carbocyclic aromatic group, such as phenyl or naphthyl, or a substituted or unsubstituted heteroaromatic group containing one or more, preferably one, heteroatom.
• alkyl and aryl groups may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent.
• Substituents may include: carbon-containing groups such as alkyl, aryl, arylalkyl (e.g. substituted and unsubstituted phenyl, substituted and unsubstituted benzyl); halogen atoms and halogen-containing groups such as haloalkyl (e.g. trifluoromethyl); oxygen-containing groups such as alcohols (e.g. hydroxyl, hydroxyalkyl, aryl(hydroxyl)alkyl), ethers (e.g.
• alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl aldehydes (e.g. carboxaldehyde), ketones (e.g. alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arycarbonylalkyl), acids (e.g. carboxy, carboxyalkyl), acid derivatives such as esters (e.g. alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl), amides (e.g.
• “Pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases.
• Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like.
• Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide.
• the chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a well known technique which is used in attempting to improve properties involving physical or chemical stability, e.g., hygroscopicity, flowability or solubility of compounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.
• “Pharmaceutically acceptable ester” refers to a conventionally esterified compound of formula I having a carboxyl group, which esters retain the biological effectiveness and properties of the compounds of formula I and are cleaved in vivo (in the organism) to the corresponding active carboxylic acid.
• ester groups which are cleaved (in this case hydrolyzed) in vivo to the corresponding carboxylic acids are those in which the cleaved hydrogen is replaced with -lower alkyl which is optionally substituted, e.g., with heterocycle, cycloalkyl, etc.
• substituted lower alkyl esters are those in which -lower alkyl is substituted with pyrrolidine, piperidine, morpholine, N-methylpiperazine, etc.
• the group which is cleaved in vivo may be, for example, ethyl, morpholino ethyl, and diethylamino ethyl.
• —CONH 2 is also considered an ester, as the —NH 2 is cleaved in vivo and replaced with a hydroxy group, to form the corresponding carboxylic acid.
• the present representative compounds may be readily synthesized by any known conventional procedure for the formation of a peptide linkage between amino acids.
• Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino group of an amino acid or residue thereof having its carboxyl group and other reactive groups protected and the free primary carboxyl group of another amino acid or residue thereof having its amino group or other reactive groups protected.
• Such conventional procedures for synthesizing the novel compounds of the present invention include for example any solid phase peptide synthesis method.
• the synthesis of the novel compounds can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principles of solid phase methods.
• Such methods are disclosed in, for example, Merrifield, R. B., J. Amer. Chem. Soc. 85, 2149-2154 (1963); Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980), which are incorporated herein by reference.
• Peptide synthesis may be performed manually or with automated instrumentation. Microwave-assisted synthesis may also be utilized.
• Alpha amino groups may be protected by a suitable protecting group selected from aromatic urethane-type protecting groups, such as allyloxycarbonyl, benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl-isopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, and allyloxycarbonyl.
• Fmoc is most preferred for alpha amino protection.
• Guanidino groups may be protected by a suitable protecting group selected from nitro, p-toluenesulfonyl (Tos), (Z,) pentamethylchromanesulfonyl (Pmc), 4-methoxy-2,3,6,-trimethylbenzenesulfonyl (Mtr). Pmc and Mtr are most preferred for arginine (Arg).
• the ⁇ -amino groups may be protected by a suitable protecting group selected from 2-chloro-benzyloxycarbonyl (2-Cl-Z),2-bromo-benzyloxycarbonyl (2-Br-Z)- and t-butyloxycarbonyl (Boc). Boc is the most preferred for (Lys).
• the ⁇ - and ⁇ -amide groups may be protected by a suitable protecting group selected from 4-methyltrityl (Mtt), 2,4,6-trimethoxybenzyl (Tmob), 4,4-dimethoxydityl/bis-(4-methoxyphenyl)-methyl (Dod) and trityl (Trt). Trt is the most preferred for (Asn) and (Gln).
• the indole group may be protected by a suitable protecting group selected from formyl (For), mesityl-2-sulfonyl (Mts) and t-butyloxycarbonyl (Boc). Boc is the most preferred for (Trp).
• ⁇ - and ⁇ -carboxyl groups may be protected by a suitable protecting group selected from t-butyl (tBu), and 2-phenylisopropyl (2Pip).
• tBu is the most preferred for (Glu) and 2Pip is most preferred for (Asp).
• the imidazole group may be protected by a suitable protecting group selected from benzyl (Bzl), t-butyloxycarbonyl (Boc), and trityl (Trt). Trt is the most preferred for (His).
• DIC Diisopropylcarbodiimide
• DIPEA diisopropylethylamine
• HOBT Hydroxybenzotriazole
• DMS dimethylsulfide
• EDT 1,2-ethanedithiol
• Protected amino acids were generally of the L configuration and were obtained commercially from Bachem, Advanced ChemTech, CEM or Neosystem. Purity of these reagents was confirmed by thin layer chromatography, NMR and melting point prior to use.
• Benzhydrylamine resin was a copolymer of styrene-1% divinylbenzene (100-200 or 200-400 mesh) obtained from Bachem, Anaspec or Advanced Chemtech. Total nitrogen content of these resins were generally between 0.3-1.2 meq/g.
• HPLC High performance liquid chromatography
• peptides were prepared using solid phase synthesis by the method generally described by Merrifield, (J. Amer. Chem. Soc., 85, 2149 (1963)), although other equivalent chemical synthesis known in the art could be used as previously mentioned.
• Solid phase synthesis is commenced from the C-terminal end of the peptide by coupling a protected alpha-amino acid to a suitable resin.
• Such a starting material can be prepared by attaching an alpha-amino-protected amino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin, or by an amide bond between an Fmoc-Linker, such as p-((R, S)- ⁇ -(1-(9H-fluoren-9-yl)-methoxyformamido)-2,4-dimethyloxybenzyl)-phenoxyacetic acid (Rink linker) to a benzhydrylamine (BHA) resin.
• Fmoc-Linker-BHA resin supports are commercially available and generally used when the desired peptide being synthesized has an unsubstituted amide at the C-terminus.
• the amino acids or mimetic are coupled onto the Fmoc-Linker-BHA resin using the Fmoc protected form of amino acid or mimetic, with 1-5 equivalents of amino acid and a suitable coupling reagent.
• the resin may be washed and dried under vacuum. Loading of the amino acid onto the resin may be determined by amino acid analysis of an aliquot of Fmoc-amino acid resin or by determination of Fmoc groups by UV analysis. Any unreacted amino groups may be capped by treating the resin with acetic anhydride and diispropylethylamine in methylene chloride or DMF.
• the resins are carried through several repetitive cycles to add amino acids sequentially.
• the alpha amino Fmoc protecting groups are removed under basic conditions.
• Piperidine, piperazine or morpholine (20-40% v/v) in DMF may be used for this purpose.
• Preferably 40% piperidine in DMF is typically utilized
• the subsequent protected amino acids are coupled stepwise in the desired order to obtain an intermediate, protected peptide-resin.
• the activating reagents used for coupling of the amino acids in the solid phase synthesis of the peptides are well known in the art.
• reagents for such syntheses are benzotriazol-1-yloxy-tri-(dimethylamino)phosphonium hexafluorophosphate (BOP), Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and diisopropylcarbodiimide (DIC). Preferred here are HBTU and DIC.
• Other activating agents are described by Barany and Merrifield (in The Peptides, Vol. 2, J.
• HOBT 1 hydroxybenzotriazole
• HOSu N-hydroxysuccinimide
• HOOBT 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
• Preferred here is HOBT.
• Protocol 1 Step Reagent Time 1 DMF 2 ⁇ 30 sec 2 20% piperidine/DMF 1 min 3 20% piperidine/DMF 15 min 4 DMF 2 ⁇ 30 sec 5 iPrOH 2 ⁇ 30 sec 6 DMF 3 ⁇ 30 sec 7 Coupling 60 min-18 hours 8 DMF 2 ⁇ 30 sec 9 iPrOH 1 ⁇ 30 sec 10 DMF 1 ⁇ 30 sec 11 CH 2 Cl 2 2 ⁇ 30 sec.
• Peptide synthesis may be performed using an Applied Biosystem 433A synthesizer (Foster City, Calif.).
• the FastMoc 0.25 mmole cycles were used with either the resin sampling or non resin sampling, 41 mL reaction vessel.
• the Fmoc-amino acid resin was dissolved with 2.1 g NMP, 2 g of 0.45M HOBT/HBTU in DMF and 2M DIEA, then transferred to the reaction vessel.
• the basic FastMoc coupling cycle was represented by the module “BADEIFD,” wherein each letter represents a module.
• B represents the module for Fmoc deprotection using 20% piperidine/NMP and related washes and readings for 30 min (either UV monitoring or conductivity);
• A represents the module for activation of amino acid in cartridges with 0.45 M HBTU/HOBt and 2.0 M DIEA and mixing with N2 bubbling;
• D represents the module for NMP washing of resin in the reaction vessel;
• E represents the module for transfer of the activated amino acid to the reaction vessel for coupling;
• I represents the module for a 10 minute waiting period with vortexing on and off of the reaction vessel; and
• F represents the module for cleaning cartridge, coupling for approximately 10 minutes and draining the reaction vessel. Couplings were typically extended by addition of module “I” once or multiple times.
• double couplings were run by performing the procedure “BADEIIADEIFD.”
• Other modules were available such as c for methylene chloride washes and “C” for capping with acetic anhydride.
• Individual modules were also modifiable by, for example, changing the timing of various functions, such as transfer time, in order to alter the amount of solvent or reagents transferred.
• the cycles above were typically used for coupling one amino acid. For synthesizing tetra peptides, however, the cycles were repeated and strung together.
• BADEIIADEIFD was used to couple the first amino acid, followed by BADEIIADEIFD to couple the second amino acid, followed by BADEIIADEIFD to couple the third amino acid, followed by BADEIIADEIFD to couple the fourth amino acid, followed by BIDDcc for final deprotection and washing.
• Peptide synthesis may be performed using a Microwave Peptide Synthesizer, Liberty (CEM Corporation, Matthews, N.C.).
• the synthesizer was programmed for double coupling and capping by modification of preloaded 0.25 mmol cycle.
• the microwave editor was used to program microwave power methods for use during the Fmoc deprotection, amino acid coupling and capping with acetic anhydride. This type of microwave control allows for methods to be created that control a reaction at a set temperature for a set amount of time.
• the Liberty automatically regulates the amount of power delivered to the reaction to keep the temperature at the set point.
• the default cycles for amino acid addition and final deprotection were selected in cycle editor and were automatically loaded while creating a peptide.
• Microwave method for deprotection 50 watts; 70° C.; 300 seconds
• Microwave method for Coupling 50 watts; 70° C.; 300 seconds
• Microwave method for Coupling 50 watts; 70° C.; 300 seconds
• Microwave Method (50 watts; 70° C.; 300 seconds)
• Fmoc-Rink-MBHA resin 1, with piperidine/DMF followed by coupling with Fmoc-AA(P) 31 with a reagent such as DIC, BOP or HBTU, where AA31 represents the 31 st amino acid residue and P represents an appropriate protecting group, yields Fmoc-AA(P) 31 -Rink-Resin, 2.
• a reagent such as DIC, BOP or HBTU
• AA31 represents the 31 st amino acid residue
• P represents an appropriate protecting group
• the blocking groups are removed and the peptide cleaved from the resin in the same step.
• the peptide-resins may be treated with 100 ⁇ L ethanedithiol, 100 ⁇ l dimethylsulfide, 300 ⁇ L anisole, and 9.5 mL trifluoroacetic acid, per gram of resin, at room temperature for 180 min.
• the peptide-resins may be treated with 1.0 mL triisopropyl silane and 9.5 mL trifluoroacetic acid, per gram of resin, at room temperature for 180 min.
• the resin is filtered off and the filtrates are precipitated in chilled ethyl ether.
• the precipitates are centrifuged and the ether layer is decanted.
• the residue was washed with two or three volumes of Et 2 O and re-centrifuged.
• the crude product 5 is dried under vacuum.
• Analogs of VIP described in the invention are agonists of the VPAC2 receptor as demonstrated in Example X. According to the elastase stability experiments in Example X, such compounds have enhanced stability to human neutrophil elastase. Therefore, administration of these VPAC2 receptor agonists would be of utility for the treatment of airway disorders such as COPD.
• the compounds of the present invention can be provided in the form of pharmaceutically acceptable salts.
• preferred salts are those formed with pharmaceutically acceptable organic acids, e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methanesulfonic, toluenesulfonic, trifluoroacetic, or pamoic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids, such as hydrohalic acids (e.g., hydrochloric acid), sulfuric acid, or phosphoric acid and the like. Any procedure for obtaining a pharmaceutically acceptable salt known to a skilled artisan can be used.
• an effective amount of any one of the peptides of this invention or a combination of any of the peptides of this invention or a pharmaceutically acceptable salt thereof is administered via any of the usual and acceptable methods known in the art, either singly or in combination.
• the compounds or compositions can thus be administered orally (e.g., buccal cavity), sublingually, parenterally (e.g., intramuscularly, intravenously, or subcutaneously), rectally (e.g., by suppositories or washings), transdermally (e.g., skin electroporation) or by inhalation (e.g., by aerosol), and in the form of solid, liquid or gaseous dosages, including tablets and suspensions.
• buccal cavity e.g., buccal cavity
• parenterally e.g., intramuscularly, intravenously, or subcutaneously
• rectally e.g., by suppositories or washings
• transdermally e.g., skin electrop
• the administration can be conducted in a single unit dosage form with continuous therapy or in a single dose therapy ad libitum.
• the therapeutic composition can also be in the form of an oil emulsion or dispersion in conjunction with a lipophilic salt such as pamoic acid, or in the form of a biodegradable sustained-release composition for subcutaneous or intramuscular administration.
• the method of the present invention is practiced when relief of symptoms is specifically required or perhaps imminent.
• the method of the present invention is effectively practiced as continuous or prophylactic treatment.
• Useful pharmaceutical carriers for the preparation of the compositions hereof can be solids, liquids or gases; thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g. binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, aerosols, and the like.
• the carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like.
• formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile.
• suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.
• compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers and the like.
• suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for proper administration to the recipient.
• the dose of a compound of the present invention depends on a number of factors, such as, for example, the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian.
• Such an amount of the active compound as determined by the attending physician or veterinarian is referred to herein, and in the claims, as an “effective amount”.
• the dose for inhalation administration is typically in the range of about 0.5 to about 100 ⁇ g/kg body weight.
• the compound of the present invention is administered at a dose rate of from about 1 ⁇ g/kg to about 50 ⁇ g/kg/day.
• Representative delivery regimens include oral, parenteral (including subcutaneous, intramuscular and intravenous), rectal, buccal (including sublingual), transdermal, pulmonary and intranasal.
• the preferred route of administration is pulmonary administration by oral inhalation.
• Methods of pulmonary administration may include aerosolization of an aqueous solution of the cyclic peptides of the present invention or the inspiration of micronized dry powder formulations. Aerosolized compositions may include the compound packaged in reverse micelles or liposomes.
• the preparation of micronized powders of suitably controlled particle size to effectively provide for alveolar delivery is well known.
• Inhalers for the delivery of specified doses of such formulations directly into the lungs are well known in the art.
• the above peptide was synthesized using Fmoc chemistry on an Applied Biosystem 433A or a microwave Peptide synthesizer.
• the synthesizer was programmed for double coupling using the modules described in Protocol 1 or 2 above.
• the synthesis was carried out on a 0.25 mmol scale using the Fmoc-Rink Linker-BHA resin (450 mg, 0.25 mmol).
• the resin was transferred to a reaction vessel on a shaker.
• the peptide resin in DMF was filtered and washed with CH 2 Cl 2 .
• the resin was treated five times with 2% TFA in CH 2 Cl 2 for 3 min each.
• the resin was immediately treated twice with 5% DIPEA/CH 2 Cl 2 and washed with CH 2 Cl 2 and DMF.
• the peptide resin was suspended in DMF in a shaker vessel securely fitted with a rubber septum. To this was added 60 mg PdCl 2 (Ph 3 P) 2 , 150 uL morpholine and 300 uL AcOH. The vessel was purged well with Ar. nBu 3 SnH was then added via syringe. The black solution was shaken for 30-45 minutes, washed with DMF and repeated. Following the second Pd treatment, the resin was washed with DMF, 2 ⁇ iPrOH, DMF, 5% DIPEA/DMF and DMF. In DMF, the peptide resin was cyclized by treatment with BOP and NMM overnight. The resin was washed with DMF and CH 2 Cl 2 and then dried under vacuum.
• the peptide was cleaved from the resin using 13.5 mL 97% TFA/3% H 2 O and 1.5 mL triisopropylsilane for 180 minutes at room temperature.
• the deprotection solution was added to 100 mL cold Et 2 O, and washed with 1 mL TFA and 30 mL cold Et 2 O to precipitate the peptide.
• the peptide was centrifuged in two 50 mL polypropylene tubes. The precipitates from the individual tubes were combined in a single tube and washed 3 times with cold Et 2 O and dried in a desiccator under house vacuum.
• the crude material was purified by preparative HPLC on a Pursuit C18-Column (250 ⁇ 50 mm, 10 ⁇ m particle size) and eluted with a linear gradient of 2-70% B (buffer A: 0.1% TFA/H 2 O; buffer B: 0.1% TFA/CH 3 CN) in 90 min., flow rate 60 mL/min, and detection 220/280 nm. The fractions were collected and were checked by analytical HPLC. Fractions containing pure product were combined and lyophilized to yield 106 mg (9.7%) of a white amorphous powder. (ES)+-LCMS m/e calculated (“calcd”) for C 159 H 256 N 46 O 47 3565.05 found 3563.7.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 28 mg (2.5%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 15.2 mg (1.4%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 79 mg (7.5%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 109 mg (10.6%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 20 mg (1.8%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 60 mg (5.3%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 40 mg (3.7%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 36 mg (3.6%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 27 mg (2.7%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 33 mg (3.3%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 55 mg (5.2%) of white amorphous powder.
• Fmoc-Rink-Linker-BHA resin (450 mg, 0.25 mmol) was subjected to solid phase synthesis and purification by following the procedure in Example 1 to yield 49 mg (4.5%) of white amorphous powder.
• the human T-lymphoid cell line Sup-Ti which expresses the VPAC2 receptor, was obtained from the American Type Culture Collection (ATCC, CRL-1942) and maintained in growth medium at densities between 0.2 and 2 ⁇ 10 6 cells/ml in a 37° C. CO 2 incubator.
• the growth medium was RPMI 1640 (Invitrogen) supplemented with 25 mM HEPES buffer and 10% fetal bovine serum (Gemini Bioproducts).
• VPAC2 agonist compound activity To evaluate VPAC2 agonist compound activity, cells in log-phase growth were washed once with growth medium at room temperature and plated into 96-well plates at a density of 4 ⁇ 10 4 cells per well in 150 uL of growth medium. Fifty uL of the compounds to be tested, prepared at appropriate concentrations in growth medium, were then added to designated wells. After 5 min at room temperature, the cells were lysed by adding 25 uL of lysis reagent 1A (cAMP Biotrak EIA system, Amersham Biosciences, RPN225) to each well. The 96-well plates were kept at room temperature for 10 min with shaking and then stored at 4° C. until analysis for cAMP (within 2 hr).
• lysis reagent 1A cAMP Biotrak EIA system, Amersham Biosciences, RPN225
• Cyclic AMP levels were determined in 100 uL of each lysate using the cAMP Biotrak Enzymeimmunoassay (EIA) kit according to the manufacture's instructions (Amersham Biosciences, RPN225).
• EIA Biotrak Enzymeimmunoassay
• the activity of each VPAC2 agonist compound was estimated by fitting the 7-concentration dose response data to a sigmoidal dose-response equation provided by the GraphPad Prism program (GraphPad Software, Inc.).
• proteolytic stabilities of peptide analogs were established with reversed phase high pressure liquid chromatography (RP HPLC) electrospray ionization mass spectrometry (ESI MS). Peptide analogs were incubated with human neutrophile elastase and the quantity of undigested analogs was determined by ESI MS at appropriate time points. Multiple peptide analogs could be included in one experiment as long as they could be differentiated by HPLC retention time and/or by molecular weight.
• RP HPLC reversed phase high pressure liquid chromatography
• ESI MS electrospray ionization mass spectrometry
• Peptide stock solutions were prepared in water to a concentration of 2.5 mg/mL. Unless in use, all stock solutions were kept at ⁇ 20° C. In order to determine the relative peptide content in the prepared stock solutions reversed phase HPLC was done with an aliquot and the observed UV absorbance was compared with a comparable aliquot from the reference standard. Concentrations of the peptide analogs were adjusted accordingly. In order to do the proteolytic digestion, peptides were dissolved in phosphate buffered saline (PBS) to a concentration of 0.1 mg/mL. As many as six different peptide analogs were mixed into one 50 ⁇ L reaction volume. The reference standard was added to all experiments as a reference and internal standard.
• PBS phosphate buffered saline
• Elastase Human Neutrophil, Calbiochem, Cat # 324681 was added from an elastase stock solution to a concentration of 1 to 2 ⁇ g/mL. Different amounts of the enzyme were chosen to compensate for the differences in the proteolytic stabilities of the peptide analogs. Previously, a stock solution of elastase was prepared in water at a concentration of 1 mg/mL. Small aliquots of the enzyme stock solution were kept at ⁇ 20° C. to better maintain the enzyme activity by limiting the number of thaw and freeze cycles.
• the digestion was done at ambient temperature in an autosampler tube within the autosampler of the HPLC system (Agilent 1100 Series). For a time course, 5 ⁇ L aliquots were injected in 70 minute intervals onto the reversed phase HPLC column (Phenomenex, Luna C18, 3 ⁇ , 100 ⁇ , 150 ⁇ 2.00 mm). For the starting time point an aliquot was injected just prior to the addition of the proteolytic enzyme. A total of eight time points could be recorded from one experiment, including the starting point. Peptides were separated on the reversed phase column with a 50 minute gradient of 5% to 30% organic phase.
• the aqueous phase was 0.05% (v/v) of trifluoroacetic acid in water and the organic one was 0.045% (v/v) of trifluoroacetic acid in acetonitrile. Absorbances were recorded at 214 and 280 nm respectively. All of the column effluent was introduced into the turbo V source of the electrospray ionization mass spectrometer (ABI 4000 QTrap LC/MS/MS System). Mass spectra were acquired in Q3MS mode in a mass range to include all triply charged ions of the non degraded peptide analogs. Care was taken to assure that peptide analogs could clearly be differentiated either by the chromatographic retention time or by the difference in molecular weight.
• Relative quantities of the respective undigested peptide analog were calculated from the integrated total ion current. A window of 2.5 Da was chosen and the manufacturer's software was used to integrate the individual ion currents. The overall halftime of an individual peptide analog was calculated by assuming first-order kinetic behavior and was normalized with respect to the halftime of the reference standard. TABLE 2 Compound in Example Relative elastase stability 1 3.9 2 5.2 3 4.5 4 3.9 5 4.9 6 6 7 16.4 8 3.5 9 12.0 10 7.4 11 2.4 12 5.2 13 1.7 14 4.5 15 4.8 16 4.4 17 4.6 18 5.1 19 3.3 20 3.4 21 5.7 22 1.8 23 3.6
• C57bl/6 mice are pretreated with vehicle or drug prior to an aerosol expose to lipopolysacchride (LPS, 500 ⁇ g/ml in sterile saline) for 15-30 minutes.
• LPS lipopolysacchride
• the aerosol is generated by a Pari Ultra neb jet nebulizer, the outlet of which is connected to a small clear plastic chamber [H ⁇ W ⁇ D, 10.7 ⁇ 25.7 ⁇ 11 cm (4 ⁇ 10 ⁇ 4.5 in)] containing the animals.
• Bronchoalveolar lavage (BAL) is performed 24 hr later to determine the intensity of cell inflammation. BAL procedure is performed as described below.
• mice are pretreated with vehicle or drug prior to an intranasal administration of lipopolysacchride (0.05-0.3 mg/kg in sterile saline; 50 ⁇ l total volume, 25 ⁇ l/nostril).
• Intranasal administration is performed by presenting small droplets of the dosing solution at the nostril using a 25-50 ⁇ l eppendorff pipet.
• BAL is performed 3 to 24 h post LPS challenged as described above to determine the intensity of cell inflammation.
• animals are anesthetized with pentobarbital (80-100 mg/kg, i.p.), ketamine/xyzaline (80-120 mg/kg/2-4 mg/kg, i.p.) or urethane (1.5-2.4 g/kg, i.p.); and through a small midline neck incision (15-20 mm), the trachea is exposed and cannulated with 20-gauge tubing adapter.
• Lungs are lavaged with 2 ⁇ 1 ml sterile Hank's balanced salt solution without Ca++ and Mg++(HBSS). Lavage fluid is recovered after 30 sec by gentle aspiration and pooled for each animal.
• Samples are then centrifuged at 2000 rpm for 10 minutes at 5° C. Supernatant is aspirated, and red blood cells are lysed from the resulting pellet with 0.5 ml distilled water for 30 sec before restoring osmolarity to the remaining cells by the addition of 5 ml of HBSS. Samples are recentrifuged at 2000 rpm for 10 minutes at 5° C. and supernatant aspirated. The resulting pellet is resuspended in 1 ml of HBSS. Total cell number is determined by Trypan Blue (Sigma Chemical, St. Louis, Mo.) exclusion from an aliquot of cell suspension using a hemocytometer or coulter counter.
• WBP whole body plethysmographs
• BUXCO Electronics, Inc. Teroy, N.Y.
• WBP chambers allow animals to move freely within the chamber while respiratory function is measured. Eight chambers are used simultaneously so that eight mice can be measured at the same time.
• Each WBP chamber is connected to a bias flow regulator to supply a smooth, constant flow of fresh air during testing.
• a transducer attached to each chamber detects pressure changes that occur as the animal breathes. Pressure signals are amplified by a MAX II Strain Gauge preamplifier and analyzed by the Biosystem XA software supplied with the system (BUXCO Electronics, Inc.).
• Pressure changes within each chamber are calibrated prior to testing by injecting exactly 1 ml of air through the injection port and adjusting the computer signal accordingly.
• Mice are placed in the WBP chambers and allowed to acclimate for 10 minutes prior to testing. Testing is conducted by letting the animals move and breathe freely for 15 minutes while the following parameters are measured: Tidal Volume (ml), Respiratory Rate (breaths per minutes), Minute Volume (tidal volume multiplied by respiratory rate, ml/min), Inspiratory Time (sec), Expiratory Time (sec), Peak Inspiratory Flow (ml/sec), and Peak Expiratory Flow (ml/sec).
• Raw data for each of the parameters listed above are captured in the software database and averaged once per minute to give a total of 15 data points per parameter. The average of the 15 data points is reported.
• Accumulated Volume (ml) is a cumulative value (not averaged) and represents the sum of all tidal volumes for the 15-minute test session.
• the protocol is customized to include measurements before, during, and after a spasmogen challenge to determine Penh. Dose-response effects of a particular spasmogen (i.e. methacholine (MCh), acetylcholine, etc.) are obtained by giving nebulized aerosol (30-60 sec exposure) at approximately 5-10 min intervals.
• MCh methacholine
• acetylcholine acetylcholine
• mice (balb/c) are treated with vehicle (2% DMSO in H 2 O) or drug dissolved in 4 ml vehicle for 20 minutes by aerosol, as described above, prior to spasmogen challenge.
• Penh is determined at 5, 30 and 60 minutes post-challenge. Data are reported as percent inhibition of Penh relative to vehicle. TABLE 4 Inhibition of Penh at 5 minutes post-challenge Compound in Example (+ >50%, ++ ⁇ 50%) 6 ++ 12 + 18 + 19 +

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